RESEARCH ARTICLE An extensive program of periodic alternative splicing linked to cell cycle progression Daniel Dominguez1,2, Yi-Hsuan Tsai1,3, Robert Weatheritt4, Yang Wang1,2, Benjamin J Blencowe4*, Zefeng Wang1,5* 1Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, United States; 2Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, United States; 3Program in Bioinformatics and Computational Biology, University of North Carolina at Chapel Hill, Chapel Hill, United States; 4Donnelly Centre and Department of Molecular Genetics, University of Toronto, Toronto, Canada; 5Key Lab of Computational Biology, CAS-MPG Partner Institute for Computational Biology, Chinese Academy of Science, Shanghai, China Abstract Progression through the mitotic cell cycle requires periodic regulation of gene function at the levels of transcription, translation, protein-protein interactions, post-translational modification and degradation. However, the role of alternative splicing (AS) in the temporal control of cell cycle is not well understood. By sequencing the human transcriptome through two continuous cell cycles, we identify ~ 1300 genes with cell cycle-dependent AS changes. These genes are significantly enriched in functions linked to cell cycle control, yet they do not significantly overlap genes subject to periodic changes in steady-state transcript levels. Many of the periodically spliced genes are controlled by the SR protein kinase CLK1, whose level undergoes cell cycle- dependent fluctuations via an auto-inhibitory circuit. Disruption of CLK1 causes pleiotropic cell cycle defects and loss of proliferation, whereas CLK1 over-expression is associated with various *For correspondence: cancers. These results thus reveal a large program of CLK1-regulated periodic AS intimately [email protected] (BJB); associated with cell cycle control. [email protected] (ZW) DOI: 10.7554/eLife.10288.001 Competing interest: See page 15 Funding: See page 16 Introduction Received: 22 July 2015 Alternative splicing (AS) is a critical step of gene regulation that greatly expands proteomic diversity. Accepted: 24 March 2016 Nearly all (>90%) human genes undergo AS and a substantial fraction of the resulting isoforms are Published: 25 March 2016 thought to have distinct functions (Pan et al., 2008; Wang et al., 2008). AS is tightly controlled, and Reviewing editor: Gene Yeo, its mis-regulation is a common cause of human diseases (Wang and Cooper, 2007). Generally, AS is University of California, San regulated by cis-acting splicing regulatory elements that recruit trans-acting splicing factors to pro- Diego, United States mote or inhibit splicing (Matera and Wang, 2014; Wang and Burge, 2008). Alterations in splicing factor expression have been observed in many cancers and are thought to activate cancer-specific Copyright Dominguez et al. splicing programs that control cell cycle progression, cellular proliferation and migration (David and This article is distributed under Manley, 2010; Oltean and Bates, 2014). Consistent with these findings, several splicing factors the terms of the Creative Commons Attribution License, function as oncogenes or tumor suppressors (Karni et al., 2007; Wang et al., 2014), and cancer- which permits unrestricted use specific splicing alterations often affect genes that function in cell cycle control (Tsai et al., 2015). and redistribution provided that Progression through the mitotic cell cycle requires periodic regulation of gene function that is pri- the original author and source are marily achieved through coordination of protein levels with specific cell cycle stages credited. (Harashima et al., 2013; Vermeulen et al., 2003). This temporal coordination enables timely control Dominguez et al. eLife 2016;5:e10288. DOI: 10.7554/eLife.10288 1 of 19 Research Article Cell biology Genomics and evolutionary biology eLife digest Mitosis is a key step in the normal life cycle of a cell, during which one cell divides into two new cells. As a cell progresses through the cell cycle, it must carefully regulate its gene activity to switch particular genes on or off at specific moments. When a gene is activated its sequence is first copied into a temporary molecule called a transcript. These transcripts are then edited to form templates to build proteins. One way that a transcript can be edited is via a process called alternative splicing, in which different pieces of the transcript are cut and pasted together to form different versions of the final template. This allows different instructions to be obtained from a single gene, introducing an added layer of biological complexity. However, the role of alternative splicing in the timing of key events of the cell life cycle is not well understood. Dominguez et al. have now looked for the genes that undergo alternative splicing during the cell cycle. The sequences of gene transcripts produced within human cells were collected while the cells went through two rounds of division. This approach revealed that around 1,300 genes are spliced in different ways at different stages of each cell cycle. Many of these genes were known to play roles in controlling the cell’s life cycle, but few of the genes showed large changes in the amount of total transcript that is generated over time. Dominguez et al. also showed that an enzyme called CLK1 influences about half of the 1,300 periodically spliced genes during the cell cycle. The production of CLK1 is itself carefully controlled throughout the cell cycle, and the enzyme’s activity prevents its own overproduction. Further experiments showed that blocking CLK1’s activity while a cell is replicating its DNA halts the cell cycle, but blocking this enzyme’s activity after the cell had replicated its DNA did not. Given this pivotal role in the cell cycle, Dominguez et al. also examined the role of CLK1 in cancer cells and found that high levels of CLK1 in tumours were linked to lower survival rates. These findings indicate that CLK1 warrants further investigation, particularly in relation to its role in cancer. DOI: 10.7554/eLife.10288.002 of molecular events that ensure accurate chromatin duplication and daughter cell segregation. Peri- odic gene function is conventionally thought to be achieved through stage-dependent gene tran- scription (Bertoli et al., 2013), translation (Grabek et al., 2015), protein-protein interactions (Satyanarayana and Kaldis, 2009), post-translational protein modifications, and ubiquitin-depen- dent protein degradation (Mocciaro and Rape, 2012). Although AS is one of the most widespread mechanisms involved in gene regulation, the relationship between the global coordination of AS and the cell cycle has not been investigated. Major families of splicing factors include the Serine-Arginine rich proteins (SR) proteins and the heterogeneous nuclear ribonucleoproteins (hnRNPs), whose levels and activities vary across cell types. SR proteins generally contain one or two RNA recognition motifs (RRMs) and a domain rich in alternating Arg and Ser residues (RS domain). Generally, RRM domains confer RNA binding specific- ity while the RS domain mediates protein-protein and protein-RNA interactions to affect splicing (Long and Caceres, 2009; Zhou and Fu, 2013). Post-translational modifications of SR proteins, most notably phosphorylation, modulate their splicing regulatory capacity by altering protein locali- zation, stability or activity (Gui et al., 1994; Lai et al., 2003; Prasad et al., 1999; Shin and Manley, 2002). Dynamic changes in SR protein phosphorylation have been detected after DNA damage (Edmond et al., 2011; Leva et al., 2012) and during the cell cycle (Gui et al., 1994; Shin and Man- ley, 2002), suggesting that regulation of AS may have important roles in cell cycle control. However, the functional consequences of SR protein (de)phosphorylation during the cell cycle are largely unclear. Through a global-scale analysis of the human transcriptome at single-nucleotide resolution through two continuous cell cycles, we have identified widespread periodic changes in AS that are coordinated with specific stages of the cell cycle. These periodic AS events belong to a set of genes that is largely separate from the set of genes periodically regulated during the cell cycle at the tran- script level, yet the AS regulated set is significantly enriched in cell cycle- associated functions. We further demonstrate that a significant fraction of the periodic AS events is regulated by the SR pro- tein kinase, CLK1, and that CLK itself is also subject to cell cycle-dependent regulation. Moreover, Dominguez et al. eLife 2016;5:e10288. DOI: 10.7554/eLife.10288 2 of 19 Research Article Cell biology Genomics and evolutionary biology inhibition or depletion of CLK1 causes pleiotropic defects in mitosis that lead to cell death or G1/S arrest, suggesting that the temporal regulation of splicing by CLK1 is critical for cell cycle progres- sion. The discovery of periodic AS thus reveals a widespread yet previously underappreciated mech- anism for the regulation of gene function during the cell cycle. Results Alternative splicing is coordinated with different cell cycle phases To systematically investigate the regulation of AS during the cell cycle, we performed an RNA-Seq analysis of synchronously dividing cells using a total of 2.3 billion reads generated across all stages (G1, S, G2 and M) of two complete rounds of the cell cycle (Figure 1—figure supplement 1A). To maximize